Hi there,

What is the program used for the fusion between the 2 PCR products? What is the overlap length? As it's hard to give you some good tips without having any details on your protocol...

It is a 21 base overlap region.  We are running a tiered assembly:

Running a standard 65C program will amplify the native gene.

Hi again,

Do you mean 65°C is the hybridization temperature for amplifying the fusion gene? If so your program looks weird as the first steps should encourage hybridization between the 2 PCR products in order to generate the full length matrix after the elongation step. But the hybridization temperature is  far less than the one for the set of primers so you encourage hybridization with primers... What you could do is to run the first few cycles at the suitable temperature for hybridization of the overlap region (and then extension at 72°C) in absence of primers and then add primers to amplify full length product only with the normal program.

I should also have stated that we have tried a normal 65°C PCR and had the same results.  I have also delayed adding the primers in case this was an issue.  I have changed the annealing temperatures, the concentrations, and delayed the addition of the primers, but all seem to give me smears.  

Would increasing annealing temperatures to 72°C work?  Or is that getting too high?

Not sure if it helps, but have you confirmed that everything that should work is definitely correct? i.e. are the sequences of the PCR products correct? (sequencing assays); primers working properly? (successful PCR amplification/ optimisation of annealing temperatures via a gradient PCR). By tiered assembly I assume that you are splicing by overlap extension? If so, primer annealing temperatures may require careful optimisation. Also, if you are using a polymerase with proofreading function, sequence errors may cause problems.

As you have rightly pointed out, may be raising the annealing temperature to 72C might be of some help. 

Too much template.  Reduce input or cut down the cycles.  If you really want to rule things out, you can take aliquots  at earlier cycles (or run multiple tubes and take them out early) and run gel to assess the number of cycles you need.

be sure that the concentration of fragmets is the same or similar...molar concentration not in nanograms......do not use too much quantity of fragmentes (in your case I will use 10 ng of big fragment)......no more tan 0.2 microM of primers.....no more tan 25-30 cycles.....and get lucky......in our lab any fragment is a different world

i routinely do fusion PCR . I first make two pcr separately, clean with kit  and use this equation for the amounts i use in fusion pcr.

ng amt of big template= (ng amount of small template) * (size of big DNA kb) / (size of small DNA kb)

normally i use maximum 300 ng of smaller template and calculate accordingly for the amount of bigger template.

I make fusion PCR without primers. So prepare standard PCR mix without primers. Set the number of cylces to 15-18 and use the annealing temperature according to fusion region, also the elongation temperature within cylce i keep one minute more than desired fragment (eg. 2kb fusion pcr product i keep 3min as elogation temperature). And you dont need to purify your DNA band from gel.

run the gel for this PCR may be load 2µL of product. You may see smear but important thing is to see if you have your desired product length. even though is looks slightly faint band.

Now use this as template for your final PCR. i use 5µL of template from fusion PCR. Set the PCR conditions exactly same as fusion PCR with primers in pcr mix.

if you have side products cut out the DNA band of interest.

I hope this helps you.

good luck.